Project 1: Development and maintenance of human glycan and phospholipid antibody repertoires Project Summary Natural antibodies (nAbs) exist in the blood of multiple mammalian species in the absence of deliberate immunization. Reactivity of nAbs with epitopes conserved between pathogens and autologous host antigens allow these antibodies to perform dual functions in immunity: providing important host defense against infection and facilitating housekeeping functions important for tissue homeostasis. The mechanisms controlling human nAb development and maintenance, however, are poorly understood. The goal of this project is to define mechanisms controlling maturation of the human natural B lymphocyte repertoire and its tissue distribution. We will achieve this goal by completing a targeted analysis of B cells reactive with conserved carbohydrate and phospholipid T lymphocyte-independent antigens associated with clinically relevant bacteria and xenoantigens. Through this approach, we will test our central hypothesis that the selection of innate-like B cell clonotypes and antigen-specific tuning of the nAb-producing B cell repertoire depend on interactions with autologous antigens and microbial antigens encountered at mucosal surfaces, which together modulate the entry of B cell clonotypes into the memory and antibody-secreting B cell compartments. In the first Specific Aim, we will sort-purify single, indexed carbohydrate- and phospholipid-binding B cells from a cohort of cadaveric human tissue donors. Immunoglobulin gene expression in these B cells will be analyzed together with expressed cellular phenotype to determine the distribution of clonal networks of nAb-producing B cells across B cell compartments in multiple human tissues. We will additionally examine the stability of innate-like B cell clonotypes and specificity of nAb repertoire by longitudinally sampling human blood after anti-CD20 (rituximab) B cell depletion, to determine the extent of antigen-reactive clonal B cell extirpation and clonal repertoire recovery during B cell compartment regeneration. Specific Aim 2 will utilize a novel, high-throughput antibody-cloning and expression platform to express immunoglobulin gene rearrangements from gene amplicon libraries as recombinant Abs and examine their binding properties, including antigen affinity and fine specificity. We will additionally examine the effects of somatic mutation on the binding properties of nAb by assessing the global reactivity of cloned antibodies and germline-reverted clonotypes using mammalian glycan antigen microarrays. This targeted analysis of antigen- reactive human B cells will permit analysis of fine antigen-specificity, affinity, and avidity of B cell clonotypes across human tissues, and the determination of whether these features of the BCR influence the tissue, B cell subset, and immunoglobulin isotype distribution of certain clones. Because nAb antigens are expressed by multiple commensal and pathogenic organisms and are protective in normal immune homeostasis, such findings will facilitate the development of immunotherapies designed to intersect the natural repertoire.